FCH 530 Homework 5
1. Go to the Protein Data Bank (PDB) webpage –
http://www.pdb.org/pdb/home/home.do
and follow the narrative for opening files.
After completing the tutorial, look up the following structures by their PDBids:
What are these proteins?
1RCP-Cytochrome c’ from Rhodobacter capsulatus
The structures of two polymorphs of cytochrome c' from Rhodobacter capsulatus (RCCP)
strain M110 have been determined by the molecular replacement method. Iron anomalous
scattering data were used to confirm the molecular replacement solution. The structures
were refined at 1.72 angstrom and 2.0 angstrom resolution to R-values of 15.0% and
16.3%, respectively. The RCCP molecule is a dimer and each of the identical 129 residue
subunits folds as a four-helical bundle with a covalently bound heme group in the center.
This structural motif resembles that of cytochromes c' reported from Rhodospirillum
molischianum (RMCP), Rhodospirillum rubrum (RRCP), Chromatium vinosum (CVCP),
Achromobacter xyloseoxidans (AXCP) and Alcaligenes denitrificans (ADCP). However,
the architecture of the RCCP dimer, that is, the mode of association of subunits, differs
substantially from that of the other cytochromes c'. In the RCCP dimer, the subunits are
roughly parallel with each other and only helix B of each subunit participates in
formation of the dimer interface. Measurement of the solvent-accessible surface area
indicates that the dimer interface is smaller in RCCP than in the other cytochromes c'. In
RMCP, CVCP, RRCP, AXCP and ADCP the subunits cross each other to form an X
shape, and two helices, A and B, of each subunit interact across the dimer interface.
These results are consistent with hydrodynamic measurements, which show that there is
an equilibrium between monomers and dimer in RCCP, whereas the dimer is the
predominant form in the other cytochromes c' for which structures have been determined.
Structural comparison of the six cytochromes c' reveal that they can be divided into two
groups. In group 1 cytochromes c', CVCP and RCCP, the amino acid sequences and the
folding of subunits are arranged in such a way as to allow the formation of a deep
channel between helices B and C with direct solvent accessibility to the heme sixth ligand
position. There is no such channel in group 2 cytochromes c', RMCP, RRCP, AXCP and
ADCP. This may account, in part, for the differences in carbon monoxide binding.
1RCB-human recombinant interleukin 4
The crystal structure of human recombinant interleukin-4 (IL-4) has been solved by
multiple isomorphous replacement, and refined to an R factor of 0.218 at 2.25 A
resolution. The molecule is a left-handed four-helix bundle with a short stretch of beta
sheet. The structure bears close resemblance to other cytokines such as granulocytemacrophage colony stimulating factor (GM-CSF). Although no sequence similarity of IL4 to GM-CSF and other related cytokines has been previously postulated, structure-based
alignment of IL-4 and GM-CSF revealed that the core of the molecules, including large
parts of all four helices and extending over half of the molecule, has 30% sequence
identity. This may have identified regions which are not only important to maintain
structure, but could also play a role in receptor binding.
Draw toplogical diagrams for each structure identifying the amino acids at the start of
new secondary structure and type of structures (see Figures 8-48 through 8-54 for
examples of topological diagrams).
1RCP
1RCB
3. Describe similarities and differences between the “lock and key,” “induced fit,”
and “transition-state analog” models.
Lock and Key
Enzyme is rigid
Induced Fit
Assumes continuous
changes in enzyme structure
due to substrate binding,
does not have preformed
binding site
Transition-state analog
active site both recognizes
and orients substrate to
activate it for reaction
Only recognizes one
substrate
Can recognize multiple
substrates
Verified by X-ray
crystallography
Can recognize multiple
substrates
Bound, distorted substrate
takes on characteristics of
transition state
4. What are the main two ways enzymes are regulated?
Enzymes are regulated by:
I. control of the amount of enzyme available. This is dependent on the rate of synthesis
of the enzyme and rate of degradation of the enzyme.
II. Control of enzyme activity by conformational or structural alterations-often through
allosterism.
5. What is allosterism? A change in the activity and conformation of an enzyme
resulting from the binding of a compound at a site on the enzyme other than the
active binding site.
Give an example of proteins and how this concept works. Allosteric regulations are
part of natural feedback control loops for feedback inhibition or feedforward
activation of enzymatic activity. An example of a protein subject to allosteric
regulation that we have looked at is the aspartate transcarbamoylase (ATCase) that
catalyzes the first step in pyrimidine biosynthesis. It is inhibited by the pyrimidine
nucleotide triphosphate CTP and activated by ATP (a purine containing nucleotide).
CTP and ATP both bind away from the active site and can cause conformational
changes in the regulatory (r) subunits of the enzyme. The activator ATP binds
preferentially to the enzyme in the active or relaxed (R) state. The inhibitor CTP
preferentially bind to the inactive state or tense (T) state of the enzyme. Binding of
the substrates causes changes in the quaternary structure that facilitate the enzyme
activity. Binding of inhibitors can hold the enzyme in a less or inactive state that is
unfavorable for the reaction to occur or for substrates to bind. Binding of activators
can hold the enzyme in an active state that is favorable for the reaction to occur or for
substrate binding.
6. What are the six classes of enzymes and what reactions do they catalyze?
1. Oxidoreductases - oxidation-reduction reactions
2. Transferases - Transfer of functional groups
3. Hydrolases - Hydrolysis reactions
4. Lyases - group elimination to form double bonds
5. Isomerases - Isomerizations (bond rearrangements)
6. Ligases - bond formation coupled with ATP
hydrolysis
7. What is the difference between a cofactor and coenzyme? Give an example of
each as they relate to biochemistry.
Cofactors are required additives for some enzymes for assisting with the catalysis of
oxidation-reduction reactions and many types of group transfer processes. They are nonprotein chemical compounds. Examples of cofactors are metal ions, coenzymes, NADH,
prosthetic groups like heme, and nucleotides.
Coenzymes are a subset of cofactors that are loosely bound to the enzyme and have
organic properties. Examples of coenzymes are vitamin-derived cofactors such as
thiamine pyrophosphate, biotin, etc.
8. For the following cofactors, what type of reactions do they help catalyze
Biotin aids in carboxylation reactions (CO2 fixation)
Cobalamine (B12) coenzymes-aids in alkylation reactions (methylation)
Coenzyme A - acyl transfer (TCA cycle)
Flavin (vitamin B2) aids in oxidation reduction reactions (nitrate reductase)
Lipoic acid - acyl transfers via oxidation reduction processes
Nicotinamide coenzymes - NAD+ independent co-substrates for redox reactions
Pyridoxal (B6) aids in amino group transfers (provides aldehyde functional group)
Tetrahydrofolate- one carbon transfers
Thiamine pyrophosphate (B1)- aids in aldehyde transfers and alpha-keto acids
decarboxylations.
Answer the following true or false. If false, explain why.
a. The initial rate of an enzyme-catalyzed reaction is independent of substrate
concentration. False. The rate, v, is independent of [S] only at levels of S>>KM
b. At saturating levels of substrate, the rate of an enzyme-catalyzed reaction is
proportional to the enzyme concentration. True. Here v = vmax = kcat[E]0
c. The Michaelis constant KM equals the substrate concentration at which v=Vmax/2.
True.
d. The KM for a regulatory enzyme varies with enzyme concentration. False. The
value of KM is independent of enzyme concentration or almost all enzymes.
e. If enough substrate is added, the normal Vmax of an enzyme-catalyzed reaction can
be attained even in the presence of a noncompetitive inhibitor. False. A
noncompetitive inhibition cannot be overcome by increasing substrate
concentration.
f. The KM of some enzymes maybe altered by the presence of metabolites
structurally unrelated to the substrate. True, these enzymes may be regulatory.
g. The rate of an enzyme-catalyzed reaction in the presence of a rate-limiting
concentration of substrate decreases with time. True, as the substrtae is used up,
the rate decreases.
h. The sigmoidal shape of the v-versus-[S] curve for some regulatory enzymes
indicates that the affinity of the enzyme for substrte de creases as the substrate
concentration is increased. False, the initial increasing slope of the curve shows
that binding of the first substrate molecules increases the affinity of the enzyme
for subsequent substrate molecules.